Immunostimulatory combinations

ABSTRACT

The present invention provides immunostimulatory combinations. Generally, the immunostimulatory combinations include a TLR agonist and a TNF/R agonist. Certain immunostimulatory combinations also may include an antigen.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/437,398, filed Dec. 30, 2002.

BACKGROUND OF THE INVENTION

There has been a major effort in recent years, with significant success,to discover new drug compounds that act by stimulating certain keyaspects of the immune system, as well as by suppressing certain otheraspects (see, e.g., U.S. Pat. Nos. 6,039,969 and 6,200,592). Thesecompounds, referred to herein as immune response modifiers (IRMs),appear to act through basic immune system mechanisms known as Toll-likereceptors (TLRs) to induce selected cytokine biosynthesis. They may beuseful for treating a wide variety of diseases and conditions. Forexample, certain IRMs may be useful for treating viral diseases (e.g.,human papilloma virus, hepatitis, herpes), neoplasias (e.g., basal cellcarcinoma, squamous cell carcinoma, actinic keratosis, melanoma), andT_(H)2-mediated diseases (e.g., asthma, allergic rhinitis, atopicdermatitis, multiple sclerosis), and are also useful as vaccineadjuvants.

Many of the IRM compounds are small organic molecule imidazoquinolineamine derivatives (see, e.g., U.S. Pat. No. 4,689,338), but a number ofother compound classes are known as well (see, e.g., U.S. Pat. Nos.5,446,153; 6,194,425; and 6,110,929) and more are still beingdiscovered. Other IRMs have higher molecular weights, such asoligonucleotides, including CpGs (see, e.g., U.S. Pat. No. 6,194,388).

In view of the great therapeutic potential for IRMs, and despite theimportant work that has already been done, there is a substantialongoing need to expand their uses and therapeutic benefits.

SUMMARY OF THE INVENTION

In one aspect, the invention provides immunostimulatory combinationsthat include a TLR agonist and a TNF/R agonist, each in an amount that,in combination with the other, is effective for increasing the immuneresponse by a subject against an antigen. In some embodiments, theimmunostimulatory combination can further include an antigen in anamount that, in combination with the other components of thecombination, is effective for inducing an immune response by a subjectagainst the antigen. In another aspect, the present invention provides amethod of inducing a T_(H)1 immune response in a subject. The methodincludes co-administering to the subject a TLR agonist and a TNF/Ragonist, each in an amount that, when in combination with the other, iseffective to induce a T_(H)1 immune response. In some embodiments, themethod further includes co-administering an antigen in an amounteffective to induce the subject to generate an immune response againstthe antigen.

In another aspect, the present invention provides a method of activatingantigen-specific CD8⁺ T cells in a subject. The method includesco-administering to the subject a TLR agonist and a TNF/R agonist, eachin an amount that, in combination with the other, is effective toactivate CD8⁺ T cells. In some embodiments, the method further includesco-administering an antigen in an amount effective to induce the subjectto generate an immune response against the antigen. In some embodiments,activating CD8⁺ cells can include expansion of CD8⁺ effector T cells. Inalternative embodiments, activating CD8⁺ T cells can include generatingCD8⁺ memory T cells.

In another aspect, the present invention provides a method of activatingantigen-specific memory CD8⁺ l T cells in a subject having priorexposure to an antigen. The method includes administering to the subjectthe antigen in an amount effective to induce antigen-specific CD8+memory T cells to become activated, thereby generating antigen-specificCD8+ effector T cells. In some embodiments, the method further includesco-administering a TLR agonist in an amount effective to induceantigen-specific CD⁺memory T cells to become activated, therebygenerating antigen-specific CD8⁺ effector T cells.

In another aspect, the present invention provides a method of treating acondition in a subject. The method includes co-administering to thesubject a TLR agonist and a TNF/R agonist, each administered in anamount that, when in combination with the other, is effective forstimulating a cell-mediated immune response. In some embodiments, themethod further includes co-administering an antigen associated with thecondition in an amount effective for inducing a cell-mediated immuneresponse.

Various other features and advantages of the present invention shouldbecome readily apparent with reference to the following detaileddescription, examples, claims and appended drawings. In several placesthroughout the specification, guidance is provided through lists ofexamples. In each instance, the recited list serves only as arepresentative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows flow cytometry data showing the results of Example 1.

FIG. 2 shows flow cytometry data showing the results of Example 2.

FIG. 3 shows flow cytometry data showing the results of Example 3.

FIG. 4 shows flow cytometry data showing the results of Example 4.

FIG. 5 shows flow cytometry data showing the results of Example 5.

FIG. 6 is a bar graph showing the results of Example 6.

FIG. 7 is a line graph showing the results of Example 7.

FIG. 8 is a bar graph showing the results of Example 8.

FIG. 9 shows flow cytometry data showing the results of Example 9.

FIG. 10A shows flow cytometry data showing the results of Example 10.

FIG. 10B is a bar graph showing the results of Example 10.

FIG. 11 shows flow cytometry data showing the results of Example 11.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The present invention provides immunostimulatory combinations andtherapeutic and/or prophylactic methods that include administering animmunostimulatory combination to a subject.

In general, the immunostimulatory combinations can provide an increasedimmune response compared to other immunostimulatory combinations and/orcompositions. Thus, methods and immunostimulatory combinations of theinvention can improve the efficacy of certain immunological treatmentsand/or provide effective treatment while using less of a component ofthe combination. This may be desirable if a particular component, whileuseful for generating a desired immunological response, is expensive,difficult to obtain, or generates undesirable side effects.

As used herein, the following terms shall have the meanings set forth:

“Agonist” refers to a compound that, in combination with a receptor, canproduce a cellular response. An agonist may be a ligand that directlybinds to the receptor. Alternatively, an agonist may combine with areceptor indirectly by, for example, (a) forming a complex with anothermolecule that directly binds to the receptor, or (b) otherwise resultingin the modification of another compound so that the other compounddirectly binds to the receptor. An agonist may be referred to as anagonist of a particular receptor or family of receptors (e.g., a TLRagonist or a TNF/R agonist).

“Antigen” refers to any substance that is capable of being the target ofan immune response. An antigen may be the target of, for example, acell-mediated and/or humoral immune response raised by a subjectorganism. Alternatively, an antigen may be the target of a cellularimmune response (e.g., immune cell maturation, production of cytokines,production of antibodies, etc.) when contacted with immune cells.

“Co-administered” refers to two or more components of a combinationadministered so that the therapeutic or prophylactic effects of thecombination can be greater than the therapeutic or prophylactic effectsof either component administered alone. Two components may beco-administered simultaneously or sequentially. Simultaneouslyco-administered components may be provided in one or more pharmaceuticalcompositions. Sequential co-administration of two or more componentsincludes cases in which the components are administered so that eachcomponent can be present at the treatment site at the same time.Alternatively, sequential co-administration of two components caninclude cases in which at least one component has been cleared from atreatment site, but at least one cellular effect of administering thecomponent (e.g., cytokine production, activation of a certain cellpopulation, etc.) persists at the treatment site until one or moreadditional components are administered to the treatment site. Thus, aco-administered combination can, in certain circumstances, includecomponents that never exist in a chemical mixture with one another.

“Immunostimulatory combination” refers to any combination of componentsthat can be co-administered to provide a therapeutic and/or prophylacticimmunostimulatory effect. The components of an immunostimulatorycombination can include, but are not limited to, TLR agonists, TNF/Ragonists, antigens, adjuvants, and the like.

“Mixture” refers to any mixture, aqueous or non-aqueous solution,suspension, emulsion, gel, cream, or the like, that contains two or morecomponents. The components may be, for example, two immunostimulatorycomponents that, together, provide an immunostimulatory combination. Theimmunostimulatory components may be any combination of one or moreantigens, one or more adjuvants, or both. For example, a mixture mayinclude two adjuvants so that the mixture forms an adjuvant combination.Alternatively, a mixture may include an adjuvant combination and anantigen so that the mixture forms a vaccine.

“Synergy” and variations thereof refer to activity (e.g.,immunostimulatory activity) of administering a combination of compoundsthat is greater than the additive activity of the compounds ifadministered individually.

“TLR” generally refers to any Toll-like receptor of any species oforganism. A specific TLR may be identified with additional reference tospecies of origin (e.g., human, murine, etc.), a particular receptor(e.g., TLR6, TLR7, TLR8, etc.), or both.

“TLR agonist” refers to a compound that acts as an agonist of a TLR.Unless otherwise indicated, reference to a TLR agonist compound caninclude the compound in any pharmaceutically acceptable form, includingany isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph,and the like. In particular, if a compound is optically active,reference to the compound can include each of the compound's enantiomersas well as racemic mixtures of the enantiomers. Also, a compound may beidentified as an agonist of one or more particular TLRs (e.g., a TLR7agonist, a TLR8 agonist, or a TLR7/8 agonist).

“TNF/R” generally refers to any member of either the Tumor NecrosisFactor (TNF) Superfamily or the Tumor Necrosis Factor Receptor (TNFR)Superfamily. The TNF Superfamily includes, for example, CD40 ligand,OX40 ligand, 4-1BB ligand, CD27, CD30 ligand (CD153), TNF-α, TNF-β, RANKligand, LT-α, LT-β, GITR ligand, and LIGHT. The TNFR Superfamilyincludes, for example, CD40, OX40, 4-1BB CD70 (CD27 ligand), CD30,TNFR2, RANK, LT-βR, HVEM, GITR, TROY, and RELT. “TNF/R agonist” refersto a compound that acts as an agonist of a member of either the TNFSuperfamily or the TNFR Superfamily. Unless otherwise indicated,reference to a TNF/R agonist compound can include the compound in anypharmaceutically acceptable form, including any isomer (e.g.,diastereomer or enantiomer), salt, solvate, polymorph, and the like. Inparticular, if a compound is optically active, reference to the compoundcan include each of the compound's enantiomers as well as racemicmixtures of the enantiomers. Also, a compound may be identified as anagonist of a particular member of either superfamily (e.g., a CD40agonist).

“Treatment site” refers to the site of a particular treatment. Dependingupon the particular treatment, the treatment site may be an entireorganism (e.g., a systemic treatment) or any portion of an organism(e.g., a localized treatment).

“Type I interferon” refers, collectively, to IFN-α, IFN-β, or anymixture or combination thereof.

“Vaccine” refers to a pharmaceutical composition that includes anantigen. A vaccine may include components in addition to the antigensuch as, for example, one or more adjuvants, a carrier, etc.

In one aspect, the invention provides immunostimulatory combinationsthat include a TLR agonist and a TNF/R agonist. Each component may, byitself, possess a certain immunostimulatory activity. In many cases, thecombination of components can provide greater immunostimulatory activitythan either component can provide alone. In certain cases, thecombination of components can provide synergistic immunostimulatoryactivity.

In certain embodiments, immunostimulatory combinations of the inventionmay be used to induce a T_(H)1 immune response in a subject to which theimmunostimulatory combination is administered. As used herein, “inducinga T_(H)1 immune response” can include instances in which theimmunostimulatory combination induces a mixed T_(H) 1/T_(H)2 response.In certain embodiments, however, the immunostimulatory combinations caninduce a T_(H)1 immune response with little or substantially noinduction of a T_(H)2 immune response.

In some embodiments, immunostimulatory combinations of the invention maybe used as an immunostimulatory adjuvant, i.e., combined with one ormore antigens, either with or without additional adjuvants. Thus, insome cases, an immunostimulatory combination may form a vaccine. Inother cases, an immunostimulatory combination may serve as an adjuvantthat may be used in connection with a vaccine.

As shown in the Examples that follow, an immunostimulatory combinationthat includes a TLR agonist and a TNF/R agonist can enhance theexpansion of activated CD8⁺ T cells, the generation of memory CD8⁺ Tcells, or both. Thus, methods and immunostimulatory combinations of theinvention can enhance antigen-specific cell-mediated immunity in asubject that receives the immunostimulatory combination or treatmentaccording to a method described in detail below.

The TLR agonist may be an agonist of any TLR desirable for a particularapplication. TLRs have been identified in various mammalian speciesincluding, for example, humans, guinea pigs, and mice. The TLR agonistmay be an agonist of any TLR (e.g., TLR6, TLR7, TLR8, etc.) from anyspecies. In some embodiments, the TLR agonist is an agonist of a humanTLR. In many cases, the TLR is a TLR from the organism to which theimmunostimulatory combination will be administered, although such acorrelation is not necessary.

Certain TLRs are known to bind certain pathogen-associated ligands. Insome cases the ligands are pathogen-derived, while in other cases theligands are subject-derived. For example, TLR3 recognizespolyinosinic-polycytidylic acid (polyIC), a “mimic” of double-strandedviral RNA; TLR4 recognizes lipopolysaccharide (LPS) of manyGram-negative bacteria; TLR5 binds certain flagellins; and TLR9 bindscertain CpG oligonucleotides. Certain small molecule IRM compounds areknown to be agonists of one or more TLRs including, for example, TLR6,TLR7, and TLR8.

In some embodiments, the TLR agonist may be an agonist of at least oneof TLR6, TLR7, TLR8, and TLR9. In certain embodiment, the TLR agonistcan be an agonist of TLR7 and/or TLR8. In alternative embodiments, theTLR agonist may be a TLR8-selective agonist. In other alternativeembodiments, the TLR agonist can be a TLR7-selective agonist.

As used herein, the term “TLR8-selective agonist” refers to any compoundthat acts as an agonist of TLR8, but does not act as an agonist of TLR7.A “TLR7-selective agonist” refers to a compound that acts as an agonistof TLR7, but does not act as an agonist of TLR8. A “TLR7/8 agonist”refers to a compound that acts as an agonist of both TLR7 and TLR8.

A TLR8-selective agonist or a TLR7-selective agonist may act as anagonist for the indicated TLR and one or more of TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR9, or TLR10. Accordingly, while “TLR8-selective agonist”may refer to a compound that acts as an agonist for TLR8 and for noother TLR, it may alternatively refer to a compound that acts as anagonist of TLR8 and, for example, TLR6. Similarly, “TLR7-selectiveagonist” may refer to a compound that acts as an agonist for TLR7 andfor no other TLR, but it may alternatively refer to a compound that actsas an agonist of TLR7 and, for example, TLR6.

The TLR agonism for a particular compound may be assessed in anysuitable manner. For example, assays for detecting TLR agonism of testcompounds are described, for example, in U.S. Provisional PatentApplication Ser. No. 60/432,650, filed Dec. 11, 2002, and recombinantcell lines suitable for use in such assays are described, for example,in U.S. Provisional Patent Application Ser. No. 60/432,651, filed Dec.11, 2002.

Regardless of the particular assay employed, a compound can beidentified as an agonist of a particular TLR if performing the assaywith a compound results in at least a threshold increase of somebiological activity mediated by the particular TLR. Conversely, acompound may be identified as not acting as an agonist of a specifiedTLR if, when used to perform an assay designed to detect biologicalactivity mediated by the specified TLR, the compound fails to elicit athreshold increase in the biological activity. Unless otherwiseindicated, an increase in biological activity refers to an increase inthe same-biological activity over that observed in an appropriatecontrol. An assay may or may not be performed in conjunction with theappropriate control. With experience, one skilled in the art may developsufficient familiarity with a particular assay (e.g., the range ofvalues observed in an appropriate control under specific assayconditions) that performing a control may not always be necessary todetermine the TLR agonism of a compound in a particular assay.

The precise threshold increase of TLR-mediated biological activity fordetermining whether a particular compound is or is not an agonist of aparticular TLR in a given assay may vary according to factors known inthe art including but not limited to the biological activity observed asthe endpoint of the assay, the method used to measure or detect theendpoint of the assay, the signal-to-noise ratio of the assay, theprecision of the assay, and whether the same assay is being used todetermine the agonism of a compound for multiple TLRs. Accordingly it isnot practical to set forth generally the threshold increase ofTLR-mediated biological activity required to identify a compound asbeing an agonist or a non-agonist of a particular TLR for all possibleassays. Those of ordinary skill in the art, however, can readilydetermine the appropriate threshold with due consideration of suchfactors.

Assays employing HEK293 cells transfected with an expressible TLRstructural gene may use a threshold of, for example, at least athree-fold increase in a TLR-mediated biological activity (e.g., NFκBactivation) when the compound is provided at a concentration of, forexample, from about 1 μM to about 10 μM for identifying a compound as anagonist of the TLR transfected into the cell. However, differentthresholds and/or different concentration ranges may be suitable incertain circumstances. Also, different thresholds may be appropriate fordifferent assays.

In certain embodiments, the TLR agonist can be a natural agonist of aTLR or a synthetic IRM compound. IRM compounds include compounds thatpossess potent immunomodulating activity including but not limited toantiviral and antitumor activity. Certain IRMs modulate the productionand secretion of cytokines. For example, certain IRM compounds inducethe production and secretion of cytokines such as, e.g., Type Iinterferons, TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12, MIP-1, and/or MCP-1.As another example, certain IRM compounds can inhibit production andsecretion of certain T_(H)2 cytokines, such as IL-4 and IL-5.Additionally, some IRM compounds are said to suppress IL-1 and TNF (U.S.Pat. No. 6,518,265).

Certain IRMs that are useful as TLR agonists in immunostimulatorycombinations of the invention are small organic molecules (e.g.,molecular weight less than about 1000 Daltons, and less than about 500Daltons in some cases), as opposed to large biological molecules such asproteins, peptides, and the like. Certain small molecule IRM compoundsare disclosed in, for example, U.S. Pat. Nos. 4,689,338; 4,929,624;4,988,815; 5,037,986; 5,175,296; 5,238,944; 5,266,575; 5,268,376;5,346,905; 5,352,784; 5,367,076; 5,389,640; 5,395,937; 5,446,153;5,482,936; 5,693,811; 5,741,908; 5,756,747; 5,939,090; 6,039,969;6,083,505; 6,110,929; 6,194,425; 6,245,776; 6,331,539; 6,376,669;6,451,810; 6,525,064; 6,545,016; 6,545,017; 6,558,951; and 6,573,273;European Patent 0 394 026; U.S. Patent Publication No. 2002/0055517; andInternational Patent Publication Nos. WO 01/74343; WO 02/46188; WO02/46189; WO 02/46190; WO 02/46191; WO 02/46192; WO 02/46193; WO02/46749 WO 02/102377; WO 03/020889; WO 03/043572 and WO 03/045391.

Additional examples of small molecule IRMs include certain purinederivatives (such as those described in U.S. Pat. Nos. 6,376,501, and6,028,076), certain imidazoquinoline amide derivatives (such as thosedescribed in U.S. Pat. No. 6,069,149), certain benzimidazole derivatives(such as those described in U.S. Pat. No. 6,387,938), and certainderivatives of a 4-aminopyrimidine fused to a five membered nitrogencontaining heterocyclic ring (such as adenine derivatives described inU.S. Pat. Nos. 6,376,501; 6,028,076 and 6,329,381; and in WO 02/085905).

Other IRMs include large biological molecules such as oligonucleotidesequences. Some IRM oligonucleotide sequences contain cytosine-guaninedinucleotides (CpG) and are described, for example, in U.S. Pat. Nos.6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705. SomeCpG-containing oligonucleotides can include synthetic immunomodulatorystructural motifs such as those described, for example, in U.S. Pat.Nos. 6,426,334 and 6,476,000. Other IRM nucleotide sequences lack CpGand are described, for example, in International Patent Publication No.WO 00/75304.

Small molecule 1RM compounds suitable for use as a TLR agonist inimmunostimulatory combinations of the invention include compounds havinga 2-aminopyridine fused to a five membered nitrogen-containingheterocyclic ring. Such compounds include, for example, imidazoquinolineamines including but not limited to substituted imidazoquinoline aminessuch as, for example, aminoalkyl-substituted imidazoquinoline amines,amide-substituted imidazoquinoline amines, sulfonamide-substitutedimidazoquinoline amines, urea-substituted imidazoquinoline amines, arylether-substituted imidazoquinoline amines, heterocyclicether-substituted imidazoquinoline amines, amido ether-substitutedimidazoquinoline amines, sulfonamido ether-substituted imidazoquinolineamines, urea-substituted imidazoquinoline ethers, andthioether-substituted imidazoquinoline amines;tetrahydroimidazoquinoline amines including but not limited toamide-substituted tetrahydroimidazoquinoline amines,sulfonamide-substituted tetrahydroimidazoquinoline amines,urea-substituted tetrahydroimidazoquinoline amines, arylether-substituted tetrahydroimidazoquinoline amines, heterocyclicether-substituted tetrahydroimidazoquinoline amines, amidoether-substituted tetrahydroimidazoquinoline amines, sulfonamidoether-substituted tetrahydroimidazoquinoline amines, urea-substitutedtetrahydroimidazoquinoline ethers, and thioether-substitutedtetrahydroimidazoquinoline amines; imidazopyridine amines including butnot limited to amide-substituted imidazopyridine amines,sulfonamido-substituted imidazopyridine amines, urea-substitutedimidazopyridine amines; aryl ether-substituted imidazopyridine amines,heterocyclic ether-substituted imidazopyridine amines, amidoether-substituted imidazopyridine amines, sulfonamido ether-substitutedimidazopyridine amines, urea-substituted imidazopyridine ethers, andthioether-substituted imidazopyridine amines; 1,2-bridgedimidazoquinoline amines; 6,7-fused cycloalkylimidazopyridine amines;imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines;oxazoloquinoline amines; thiazoloquinoline amines; oxazolopyridineamines; thiazolopyridine amines; oxazolonaphthyridine amines; andthiazolonaphthyridine amines.

In certain embodiments, the TLR agonist may be an imidazonaphthyridineamine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinolineamine, a thiazoloquinoline amine, an oxazolopyridine amine, athiazolopyridine amine, an oxazolonaphthyridine amine, or athiazolonaphthyridine amine.

In certain embodiments, the TLR agonist can be a sulfonamide-substitutedimidazoquinoline amine. In alternative embodiments, the TLR agonist canbe a urea-substituted imidazoquinoline ether. In another alternativeembodiment, the TLR agonist can be an aminoalkyl-substitutedimidazoquinoline amine.

In one particular embodiment, the TLR agonist is4-amino-α,α,2-trimethyl-1H -imidazo[4,5-c]quinolin-1-ethanol. In analternative particular embodiment, the TLR agonist isN-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-N-methylmorpholine-4-carboxamide.In another alternative embodiment, the TLR agonist is1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine. In another alternative embodiment, theTLR agonist isN-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide.In yet another alternative embodiment, the TLR agonist isN-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide.

In certain alternative embodiments, the TLR agonist may be a substitutedimidazoquinoline amine, a tetrahydroimidazoquinoline amine, animidazopyridine amine, a 1,2-bridged imidazoquinoline amine, a 6,7-fusedcycloalkylimidazopyridine amine, an imidazonaphthyridine amine, atetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, athiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridineamine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.

As used herein, a substituted imidazoquinoline amine refers to anaminoalkyl -substituted imidazoquinoline amine, an amide-substitutedimidazoquinoline amine, a sulfonamide-substituted imidazoquinolineamine, a urea-substituted imidazoquinoline amine, an arylether-substituted imidazoquinoline amine, a heterocyclic ether-substituted imidazoquinoline amine, an amido ether-substitutedimidazoquinoline amine, a sulfonamido ether-substituted imidazoquinolineamine, a urea-substituted imidazoquinoline ether, or athioether-substituted imidazoquinoline amines. As used herein,substituted imidazoquinoline amines specifically and expressly exclude1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine and4-amino-α,α-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-l-ethanol.

The TNF/R agonist may be any suitable agonist of any member of eitherthe TNF Superfamily or the TNFR Superfamily. In many cases, a member ofone Superfamily can be an agonist of a complementary member of the otherSuperfamily. For example, CD40 ligand (a member of the TNF Superfamily)can act as an agonist of CD40 (a member of the TNFR Superfamily), andCD40 can act as an agonist of CD40 ligand. Thus, suitable TNF/R agonistsinclude, for example, CD40 ligand, OX40 ligand, 4-1BB ligand, CD27, CD30ligand (CD153), TNF-α, TNF-β, RANK ligand, LT-α, LT-β, GITR ligand,LIGHT, CD40, OX40, 4-1BB, CD70 (CD27 ligand), CD30, TNFR2, RANK, LT-βR,HVEM, GITR, TROY, and RELT. Additionally, suitable TNF/R agonistsinclude certain agonistic antibodies raised against a TNF/R (e.g., 1C10and FGK4.5, each of which was raised against mouse CD40).

The TLR agonist and TNF/R agonist are provided (or administered, asappropriate to the form of the immunostimulatory combination) in anamount effective to increase the immune response to a particularantigen. For example, the TLR agonist can be administered in an amountfrom about 100 ng/kg to about 100 mg/kg. In many embodiments, the TLRagonist is administered in an amount from about 10 μg/kg to about 10mg/kg. In some embodiments, the TLR agonist is administered in an amountfrom about 1 mg/kg to about 5 mg/kg. The particular amount of TLRagonist that constitutes an amount effective to increase the immuneresponse to a particular antigen, however, depends to some extent uponcertain factors including but not limited to the particular TLR agonistbeing administered; the particular antigen being administered and theamount thereof; the particular TNF/R agonist being administered and theamount thereof; the state of the immune system (e.g., suppressed,compromised, stimulated); the method and order of administration of theTLR agonist, the TNF/R agonist, and the antigen; the species to whichthe formulation is being administered; and the desired therapeuticresult. Accordingly it is not practical to set forth generally theamount that constitutes an effective amount of the TLR agonist. Those ofordinary skill in the art, however, can readily determine theappropriate amount with due consideration of such factors.

Also, for example, the TNF/R agonist may be administered in an amountfrom about 100 ng/kg to about 100 mg/kg. In certain embodiments, theTNF/R agonist is administered in an amount from about 10 μg/kg to about10 mg/kg. In some embodiments, the TNF/R agonist is administered in anamount from about 1 mg/kg to about 5 mg/kg. The particular amount ofTNF/R agonist that constitutes an amount effective to increase theimmune response to a particular antigen, however, depends to some extentupon certain factors including but not limited to the particular TNF/Ragonist being administered; the particular TLR agonist beingadministered and the amount thereof; the particular antigen beingadministered and the amount thereof; the state of the immune system; themethod and order of administration of the TLR agonist, the TNF/Ragonist, and the antigen; the species to which the formulation is beingadministered; and the desired therapeutic result. Accordingly it is notpractical to set forth generally the amount that constitutes aneffective amount of the TNF/R agonist. Those of ordinary skill in theart, however, can readily determine the appropriate amount with dueconsideration of such factors.

In some embodiments, the immunostimulatory combination may furtherinclude an antigen. When present in the immunostimulatory combination,the antigen may be administered in an amount that, in combination withthe other components of the combination, is effective to generate animmune response against the antigen. For example, the antigen can beadministered in an amount from about 100 ng/kg to about 100 mg/kg. Inmany embodiments, the antigen may be administered in an amount fromabout 10 μg/kg to about 10 mg/kg. In some embodiments, the antigen maybe administered in an amount from about 1 mg/kg to about 5 mg/kg. Theparticular amount of antigen that constitutes an amount effective togenerate an immune response, however, depends to some extent uponcertain factors such as, for example, the particular antigen beingadministered; the particular TLR agonist being administered and theamount thereof; the particular TNF/R agonist being administered and theamount thereof; the state of the immune system; the method and order ofadministration of the TLR agonist, the TNF/R agonist, and the antigen;the species to which the formulation is being administered; and thedesired therapeutic result. Accordingly, it is not practical to setforth generally the amount that constitutes an effective amount of theantigen. Those of ordinary skill in the art, however, can readilydetermine the appropriate amount with due consideration of such factors.

When present, the antigen may be administered simultaneously orsequentially with any component of the immunostimulatory combination.Thus, the antigen may be administered alone or in a mixture with one ormore adjuvants (including, e.g., a TLR agonist, a TNF/R agonist, orboth). In some embodiments, an antigen may be administeredsimultaneously (e.g., in a mixture) with respect to one adjuvant, butsequentially with respect to one or more additional adjuvants.

Sequential co-administration of an antigen and other components of animmunostimulatory combination can include cases in which the antigen andat least one other component of the immunostimulatory combination areadministered so that each is present at the treatment site at the sametime, even though the antigen and the other component are notadministered simultaneously. Sequential co-administration of the antigenand the other components of the immunostimulatory combination also caninclude cases in which the antigen or at least one of the othercomponents of the immunostimulatory combination is cleared from atreatment site, but at least one cellular effect of the cleared antigenor other component (e.g., cytokine production, activation of a certaincell population, etc.) persists at the treatment site at least until oneor more additional components of the combination are administered to thetreatment site. Thus, it may be possible that an immunostimulatorycombination of the invention can, in certain circumstances, include oneor more components that never exist in a mixture with another componentof the combination.

The antigen can be any material capable of raising a T_(H)1 immuneresponse, which may include one or more of, for example, a CD8⁺ T cellresponse, an NK T cell response, a γ/δ T cell response, or a T_(H)1antibody response. Suitable antigens include but are not limited topeptides; polypeptides; lipids; glycolipids; polysaccharides;carbohydrates; polynucleotides; prions; live or inactivated bacteria,viruses or fungi; and bacterial, viral, fungal, protozoal,tumor-derived, or organism-derived antigens, toxins or toxoids.

Furthermore, it is contemplated that certain currently experimentalantigens, especially materials such as recombinant proteins,glycoproteins, and peptides that do not raise a strong immune response,can be used in connection with adjuvant combinations of the invention.Exemplary experimental subunit antigens include those related to viraldisease such as adenovirus, AIDS, chicken pox, cytomegalovirus, dengue,feline leukemia, fowl plague, hepatitis A, hepatitis B, HSV-1, HSV-2,hog cholera, influenza A, influenza B, Japanese encephalitis, measles,parainfluenza, rabies, respiratory syncytial virus, rotavirus, wart, andyellow fever.

In certain embodiments, the antigen may be a cancer antigen or a tumorantigen. The terms cancer antigen and tumor antigen are usedinterchangeably and refer to an antigen that is differentially expressedby cancer cells. Therefore, cancer antigens can be exploited todifferentially target an immune response against cancer cells. Cancerantigens may thus potentially stimulate tumor-specific immune responses.Certain cancer antigens are encoded, though not necessarily expressed,by normal cells. Some of these antigens may be characterized as normallysilent (i.e., not expressed) in normal cells, those that are expressedonly at certain stages of differentiation, and those that are temporallyexpressed (e.g., embryonic and fetal antigens). Other cancer antigenscan be encoded by mutant cellular genes such as, for example, oncogenes(e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), orfusion proteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried by RNA and DNA tumor viruses.

Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its antigenicepitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA)and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-ζ chain, MAGE-family oftumor antigens (e.g., MAGE-AL MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin,α-catenin, β-catenin, γ-catenin, p120ctn, gp100^(Pmel117), PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig -idiotype, p15, gp75, GM2 and GD2 gangliosides, viralproducts such as human papilloma virus proteins, Smad family of tumorantigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL -40), SSX-3, SSX-4, SSX-5,SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and specific tumor antigens associated with suchtumors (but not exclusively), include acute lymphoblastic leukemia(etv6, aml1, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma(E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn), bladder cancer(p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu,c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras,HER2/neu, c-erbB-2, MUC family), colorectal cancer (Colorectalassociated antigen (CRC)-0017-1A/GA733, APC), choriocarcinoma (CEA),epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu,c-erbB-2, ga733 glycoprotein), hepatocellular cancer (α-fetoprotein),Hodgkins lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1),lymphoid cell-derived leukemia (cyclophilin b), melanoma (p15 protein,gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1,cdc27, MAGE-3, p21ras, gp100^(Pmel117)), myeloma (MUC family, p21ras),non-small cell lung carcinoma (HER2/neu, c-erbB-2), nasopharyngealcancer (Imp-1, EBNA-1), ovarian cancer (MUC family, HER2/neu, c-erbB-2),prostate cancer (Prostate Specific Antigen (PSA) and its antigenicepitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733glycoprotein), renal cancer (HER2/neu, c-erbB-2), squamous cell cancersof the cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), and T cell leukemia(HTLV-1 epitopes).

Immunostimulatory combinations of the invention that include an antigenmay form a vaccine. Such vaccines can contain additionalpharmaceutically acceptable ingredients, excipients, carriers, and thelike well known to those skilled in the art.

Immunostimulatory combinations of the invention can be administered toanimals, e.g., mammals (human and non-human), fowl, and the likeaccording to conventional methods well known to those skilled in the art(e.g., orally, subcutaneously, nasally, topically).

The invention also provides therapeutic and/or prophylactic methods thatinclude administering an immunostimulatory combination of the inventionto a subject.

Unless a specific sequence of administration is provided, components ofthe immunostimulatory combination may be administered simultaneouslywith the antigen (together in admixture or separately, e.g., orally orby separate injection) or subsequent to administering one or more othercomponents of the immunostimulatory combination. For example, a TLRagonist and a TNF/R agonist may be administered simultaneously with oneanother or sequentially with respect to each other. Also, when anantigen is present as a component of the immunostimulatory combination,it may be administered simultaneously with, or sequentially with respectto, any other component of the combination.

Components of the immunostimulatory combination can be administeredsimultaneously or sequentially in any order. When the components areadministered simultaneously, they can be administered in a singleformulation or in distinct formulations. When administered as distinctformulations, whether simultaneously or sequentially, the components maybe administered at a single site or at separate sites. Also, whenadministered as distinct formulations, each formulation may beadministered using a different route. Suitable routes of administrationinclude but are not limited to transdermal or transmucosal absorption,injection (e.g., subcutaneous, intraperitoneal, intramuscular,intravenous, etc.), ingestion, inhalation, and the like. Whenadministered sequentially, the time between administration of thecomponents can be determined, at least in part, by certain factors suchas, for example, the length of time a particular component persists,either systemically or at the administration site; or the length of timethat the cellular effects of the component persist, either systemicallyor at the administration site, even after the component has beencleared.

Certain small molecule IRM compounds can induce biosynthesis ofantiviral cytokines. Therefore, for certain embodiments that include alive viral antigen and a small molecule IRM compound as the TLR agonistcomponent of the immunostimulatory combination, it may be desirable toadminister the antigen prior to administering the IRM compound so thatthe viral infection can be established.

In one aspect, methods of the invention can include administering avaccine including an immunostimulatory combination of the invention toinduce a T_(H)1 immune response in a subject. As noted above, certainsmall molecule IRMs, alone, may be useful as a vaccine adjuvant. Animmunostimulatory combination that includes a TLR agonist (e.g., a smallmolecule IRM) and a TNF/R agonist can provide an even greater immuneresponse than either an antigen alone, an antigen combined with a TLRagonist, or an antigen combined with a TNF/R agonist. In some cases, animmunostimulatory combination that includes a TLR agonist and a TNF/Ragonist can synergistically increase an immune response compared toeither a TLR agonist or TNF/R agonist.

Methods of the invention also include inducing an immune response fromcells of the immune system regardless of whether the cells are in vivoor ex vivo. Thus, an immunostimulatory combination of the invention maybe useful as a component of a therapeutic vaccine, a component of aprophylactic vaccine, or as an immunostimulatory factor used in ex vivocell culture. When used to elicit an immune response ex vivo, the immunecells activated ex vivo may be reintroduced into a patient.Alternatively, factors secreted by the activated immune cells in thecell culture, (e.g., antibodies, cytokines, co-stimulatory factors, andthe like) may be collected for investigative, prophylactic, ortherapeutic uses.

Methods of the invention also include activating naive CD8⁺ T cells inan antigen-specific manner in vivo. The population of activatedantigen-specific CD8⁺ l T cells produced in response toco-administration of an antigen and an immunostimulatorycombination—whether or not the antigen is explicitly a component of theimmunostimulatory combination—may be divided into two functionallydistinct sub-populations. One population of antigen-specific CD8⁺ Tcells includes effector T cells,—CD8⁺ T cells actively engaged inproviding a cell-mediated immune response. A second population ofantigen-specific CD8⁺ T cells includes memory T cells, CD8⁺ T cells thatare not themselves involved in providing an immune response, but can bereadily induced to become antigen-specific effector cells upon a latercontact with the same antigen. Activation of CD8⁺ T cells according tothe following method may induce expansion of antigen-specific CD8⁺effector T cells, generate antigen-specific CD8⁺ memory T cells, orboth.

An immunostimulatory combination that includes an antigen may beadministered to a subject. After sufficient incubation in the subject,CD8⁺ T cells will mature to antigen-specific CD8⁺ effector T cells inresponse to the immunization. A greater percentage of CD8⁺ effector Tcells will be antigen-specific in subjects immunized with animmunostimulatory combination that includes a TLR agonist and a TNF/Ragonist compared to subjects immunized with only antigen, antigen and aTNF/R agonist, or antigen and a TLR agonist. FIG. 1 shows flow cytometrydata demonstrating the increased expansion of antigen-specific CD8⁺effector T cells when a subject is immunized with an immunostimulatorycombination of the invention.

Generally, the incubation time between immunization and the generationof CD8⁺ effector T cells is from about 4 days to about 12 days. Incertain embodiments, CD8⁺effector T cells may be generated in about 5days after immunization. In other embodiments, CD8⁺ effector T cells maybe generated in about 7 days after immunization.

If the antigen is a protein, it may not be necessary to administer theentire protein to the subject. FIG. 2 shows expansion kinetics of CD8⁺ Tcells in response to whole chicken ovalbumin, but FIG. 1 shows expansionof CD8⁺ T cells using an eight amino acid peptide from chicken ovalbumin(SIINFEKL, SEQ ID NO:1). Similarly, FIG. 3 shows expansion of CD8⁺ Tcells in response to a TRP2-ΔV peptide (SIYDFFVWL, SEQ ID NO:2).

Thus, a method that includes administering to a subject animmunostimulatory combination of the invention may be used to elicit anantigen-specific response in CD8⁺ cytotoxic T lymphocytes (CTLs) of thesubject. Such a response may be directed against many conditionsincluding, for example, tumors and virus-infected cell populations. Insome embodiments of the invention, a vaccine of the invention may beadministered prophylactically to provide a subject with a protectiveantigen-specific cell-mediated immunity directed against, for example,tumors and/or viral infections.

In an alternative embodiment, immunostimulatory combinations of thepresent invention may be used to develop antigen-specific CD8⁺ memory Tcells in vivo. The antigen-specific CD8⁺ memory T cells may be capableof generating a secondary T_(H)1 immune response upon a second exposureto the antigen. CD8⁺ l effector T cells may be generated from there-activated CD8⁺ memory T cells in as little as 2 hours afterre-exposure to the antigen. The second exposure to the antigen may be byimmunization (i.e., a booster immunization) or natural exposure.

FIG. 4 shows re-activation of antigen-specific CD8⁺memory T cells fourweeks after being generated by co-administration of an antigen, a TLRagonist, and a TNF/R agonist. Re-activation of the CD8⁺ memory T cellsis induced by challenge with an antigen (panel B), but is even greaterwhen challenged with co-administered antigen and TLR agonist (panel C).In certain cases, the antigen-specific cell-mediated immunologic memorydescribed above may be supplemented by antigen-specific humoralimmunologic memory provided by circulating antibodies resulting from aT_(H)2 immune response to one or more components of a vaccine.

An immunostimulatory combination of the invention can be used totherapeutically treat a condition treatable by a cell-mediated immuneresponse. Such a combination can contain at least a therapeuticallyeffective amount of a TLR agonist and a therapeutically effective amountof a TNF/R agonist. In many embodiments, a therapeutic combination canfurther include a therapeutically effective amount of an antigen.

A therapeutic combination can be provided in further combination withone or more pharmaceutically acceptable carriers. Because the TLRagonist, TNF/R agonist, and antigen (if present in the combination) maybe co-administered sequentially, at different sites, and/or by differentroutes, a therapeutic combination may be provided in two or moreformulations. When provided in two or more formulations, eachformulation can include a carrier similar or different than the carrieror carriers included in the remaining formulations. Alternatively, theTLR agonist, TNF/R agonist, and antigen (if present in the combination)may be provided in a single formulation, which can include a singlecarrier or a combination of carriers.

Each component or mixture of components may be administered in anysuitable conventional dosage form such as, for example, tablets,lozenges, parenteral formulations, syrups, creams, ointments, aerosolformulations, transdermal patches, transmucosal patches and the like.

Therapeutic immunostimulatory combinations can be administered as thesingle therapeutic agent in the treatment regimen. Alternatively, atherapeutic immunostimulatory combination of the invention may beadministered in combination with another therapeutic combination of theinvention, with one or more pharmaceutical compositions, or with otheractive agents such as antivirals, antibiotics, additional IRM compounds,etc.

Because of their ability to induce the T_(H)1 immune response andgenerate a pool of CD8⁺ effector T cells, certain immunostimulatorycombinations of the invention can be particularly useful for treatingviral diseases and tumors. This immunomodulating activity suggests thatimmunostimulatory combinations and vaccines of the invention are usefulin treating conditions such as, but not limited to:

(a) viral diseases such as, for example, diseases resulting frominfection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, orVZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, ormolluscum contagiosum), a picornavirus (e.g., rhinovirus orenterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus(e.g., parainfluenzavirus, mumps virus, measles virus, and respiratorysyncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g.,papillomaviruses, such as those that cause genital warts, common warts,or plantar warts), a hepadnavirus (e.g., hepatitis B virus), aflavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus(e.g., a lentivirus such as HIV);

(b) bacterial diseases such as, for example, diseases resulting frominfection by bacteria of, for example, the genus Escherichia,Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria,Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas,Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria,Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter,Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus,or Bordetella;

(c) other infectious diseases, such chlamydia, fungal diseases includingbut not limited to candidiasis, aspergillosis, histoplasmosis,cryptococcal meningitis, or parasitic diseases including but not limitedto malaria, pneumocystis carnii pneumonia, leishmaniasis,cryptosporidiosis, toxoplasmosis, and trypanosome infection; and

(d) neoplastic diseases, such as, for example, intraepithelialneoplasias, cervical dysplasia, actinic keratosis, basal cell carcinoma,squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma,melanoma, renal cell carcinoma, leukemias including but not limited tomyelogeous leukemia, chronic lymphocytic leukemia, multiple myeloma,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-cell lymphoma, andhairy cell leukemia, and other cancers (e.g., cancers identified above);and

(e) T_(H)2-mediated, atopic, and autoimmune diseases, such as atopicdermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis,systemic lupus erythematosus, essential thrombocythaemia, multiplesclerosis, Ommen's syndrome, discoid lupus, alopecia areata, inhibitionof keloid formation and other types of scarring, and enhancing wouldhealing, including chronic wounds.

Some embodiments of the immunostimulatory combinations of the inventionalso may be useful as a vaccine adjuvant for use in conjunction with anymaterial that raises either humoral and/or cell mediated immuneresponse, such as, for example, live viral, bacterial, or parasiticantigens; inactivated viral, tumor-derived, protozoal, organism-derived,fungal, or bacterial antigens, toxoids, toxins; self-antigens;polysaccharides; proteins; glycoproteins; peptides; cellular vaccines;DNA vaccines; recombinant proteins; glycoproteins; peptides; and thelike, for use in connection with, for example, BCG, cholera, plague,typhoid, hepatitis A, hepatitis B, hepatitis C, influenza A, influenzaB, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever,tetanus, diphtheria, hemophilus influenza b, tuberculosis, meningococcaland pneumococcal vaccines, adenovirus, HIV, chicken pox,cytomegalovirus, dengue, feline leukemia, fowl plague, HSV-1 and HSV-2,hog cholera, Japanese encephalitis, respiratory syncytial virus,rotavirus, papilloma virus, yellow fever, and Alzheimer's Disease.

Immunostimulatory combinations of the invention may also be particularlyhelpful in individuals having compromised immune function. For example,IRM, compounds may be used for treating the opportunistic infections andtumors that occur after suppression of cell mediated immunity in, forexample, transplant patients, cancer patients and HIV patients.

The invention also provides a method of treating a viral infection in ananimal and a method of treating a neoplastic disease in an animalcomprising administering a therapeutically effective amount of animmunostimulatory combination of the invention to the animal. Atherapeutically effective amount to treat or inhibit a viral infectionis an amount that will cause a reduction in one or more of themanifestations of viral infection, such as viral lesions, viral load,rate of virus production, and mortality as compared to untreated controlanimals. A therapeutically effective amount of a combination to treat aneoplastic condition is an amount that will cause, for example, areduction in tumor size, a reduction in the number of tumor foci, orslow the growth of a tumor, as compared to untreated animals.

In one particular embodiment, an immunostimulatory combination of theinvention may be used to inhibit tumor growth in vivo. Subjects havingtumor cells expressing a particular antigen may be immunized with atherapeutic combination that contains a TLR agonist, a TNF/R agonist,and, optionally, the antigen. In some embodiments, the therapy caninclude an initial immunization and a second booster immunization.Tumors taken from subjects immunized with a therapeutic combination ofthe invention were generally smaller than the tumors harvested fromeither (a) non-immunized subjects, or (b) subjects immunized with onlythe antigen (FIGS. 5 and 6).

FIG. 6 compares tumor size in mice challenged with melanoma cells thatexpress ovalbumin as a tumor antigen. Seven days after challenge withthe melanoma cells, the mice were immunized with either (a) ovalbuminpeptide, (b) ovalbumin peptide and TLR agonist, or (c) ovalbuminpeptide, TLR agonist, and TFNR agonist. On day 21 (14 days afterimmunization), tumors were removed and measured. The antigen/TLRagonist/TFNR agonist combination provided superior protection againsttumor growth compared to the protection provided by immunization withthe antigen or an antigen/TLR agonist combination.

FIG. 7 compares tumor size in mice challenged with melanoma cells thatexpress ovalbumin as a tumor antigen, in which (a) the mice received twoimmunizations against the tumor, and (b) the antigen component of theimmunization included tumor cell lysate rather than ovalbumin peptide.FIG. 7 shows that immunization with a combination of TNF/R agonist andantigen provided little or no protection against tumor growth comparedto mice immunized with only antigen. Again, the antigen/TLR agonist/TFNRagonist combination provided superior protection against tumor growthcompared to the protection provided by immunization with the antigen oran antigen/TLR agonist combination.

In some cases, the extent to which the synergistic nature of an immuneresponse to an immunostimulatory combination depends upon Type Iinterferon correlates with the Type I interferon stimulation typicallyobserved by activating the TLR that is activated by the TLR agonist ofthe combination. FIG. 10 shows that the synergistic nature of an immuneresponse to an immunostimulatory combination that includes, as the TLRagonist, an agonist of a TLR that typically induces Type I interferons(e.g., TLR7, TLR3, TLR9, and TLR4) can be significantly reduced in micelacking receptors for Type I interferons. Thus, the synergistic immuneresponse to such immunostimulatory combinations is at least partiallydependent upon Type I interferon.

FIG. 10 also shows, however, that the synergistic immune responsegenerated with an immunostimulatory combination that includes an agonistof a TLR that typically induces very little or no Type I interferonsynthesis (MALP-2, a TLR2/6 agonist) is independent of Type Iinterferon.

Furthermore, FIG. 11 shows that the interferon-independent synergisticimmune response induced by an immunostimulatory combination thatincludes MALP-2 can be induced using other TLR2 agonists. For example,the TLR2 agonist Pam3cys also can induce a synergistic immune responsein IFNαβ receptor knock out mice (i.e., mice unable to processinterferon-dependent cellular signal).

Thus, it may be possible, using the methods of the invention, to tailoran immunostimulatory combination according to a desired level of Type Iinterferon induction, a desired Type I interferon dependency of theimmune response, or both. For example, an immunostimulatory combinationthat includes a TLR7 agonist may be desirable when a high level ofinterferon induction and/or an immune response that is Type I interferondependent is sought such as, for example, for providing therapeutic orprophylactic treatment against a viral infection. Alternatively, forcases in which a synergistic immune response is sought without inducingType I interferon production, an immunostimulatory combination mayinclude a TLR2 agonist such as, for example, for providing therapeuticor prophylactic treatment against a subcutaneous bacterial infection ora parasitic infection.

Treatments according to the present invention may include one or morethan one immunization. When the treatment includes more than oneimmunization, the treatment can include any suitable number ofimmunizations administered at any suitable frequency. The number andfrequency of immunizations in a treatment regimen depend at least inpart upon one or more factors including but not limited to the conditionbeing treated and the stage thereof, the state of the subject's immunesystem, the particular TLR agonist being administered and the amountthereof, the particular TNF/R agonist being administered and the amountthereof, and the particular antigen being administered (if present) andthe amount thereof.

In some embodiments, therapeutic combinations of the invention may notrequire an antigen component. For certain conditions (e.g., B celllymphoma or chronic bacterial or viral infections), effective treatmentmay be obtained using an immunostimulatory combination that does notinclude an antigen. Such conditions may be treatable in this waybecause, for example, the condition may provide a sufficient quantity orvariety of condition-specific antigens to generate a cell-mediatedimmune response capable of treating the condition.

EXAMPLES

The following examples have been selected merely to further illustratefeatures, advantages, and other details of the invention. It is to beexpressly understood, however, that while the examples serve thispurpose, the particular materials and amounts used as well as otherconditions and details are not to be construed in a matter that wouldunduly limit the scope of this invention.

Unless otherwise indicated, mice used in the following examples areC57BL6 mice, available from Charles River Laboratories, Inc.,Wilmington, Mass.

TLR agonists used in the Examples that follow are identified in Table 1.

TABLE 1 TLR agonist Compound Name Reference IRM14-amino-α,α,2-trimethyl-1H-imidazo[4,5- U.S. Pat. No.c]quinolin-1-ethanol 5,266,575 Example C1 IRM2N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H- WO 02/46191imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)- Example 6N-methylmorpholine-4-carboxamide IRM31-(2-amino-2-methylpropyl)-2-(ethoxy- U.S. Pat. No.methyl)-1H-imidazo[4,5-c]quinolin-4-amine 6,069,149^(#) IRM4N-[4-(4-amino-2-ethyl-1H-imidazo[4,5- U.S. Pat. No.c]quinolin-1-yl)butyl]-methanesulfonamide 6,331,539^(#) IRM5N-[4-(4-amino-2-propyl-1H-imidazo[4,5- U.S. Pat. No.c]quinolin-1-yl)butyl]-methanesulfonamide 6,331,539^(#) ^(#)Thiscompound is not specifically exemplified but can be readily preparedusing the synthetic methods disclosed in the cited reference.

Ovalbumin peptide (SHNFEKL, SEQ ID NO:1) and TRP2-ΔV peptide (SIYDFFVWL,SEQ ID NO:2) were obtained from American Peptide Co., Sunnyvale, Calif.

MHC tetrameric reagent was prepared using a eukaryotic (Baculovirus)expression system as follows/described in Kedl et al., JEM192(8):1105-1113 (2000).

Example 1

2-5 Mice were immunized intravenously with (A) 100 μg ovalbumin peptide,(B) 100 μg ovalbumin peptide+100 μg anti-CD40 antibody (1C10), (C) 100μg ovalbumin peptide+200 μg IRM1, or (D) 100 μg ovalbumin peptide+100 μgIC10 anti-CD40 antibody+200 μg IRM1. At five days after theimmunizations, the spleens were removed from the mice and homogenized.The homogenized cell suspension was stained with a majorhistocompatibility complex (MHC) tetrameric reagent for detectingovalbumin-specific T cells (Kedl et al., JEM 192(8):1105-1113 (2000)), aCD8 stain (BD Biosciences Pharmingen, San Diego, Calif.), and a CD44stain (BD Biosciences Pharmingen, San Diego, Calif. When subjected toflow cytometry, ovalbumin-specific CD8⁺ T cells are shown in the upperright quadrant of the dot plots shown in FIG. 1. Expansion of theovalbumin-specific CD8⁺ T cell population after stimulation with thecombination of the anti-CD40 antibody and IRM was greater than theexpansion of the ovalbumin-specific CD8⁺ T cell populations afterstimulation with either the anti-CD40 antibody or IRM alone.

Example 2

Mice were intraperitoneally injected with 5 mg ovalbumin (Sigma ChemicalCo., St. Louis, Mo.), 50 μg FGK4.5 anti-CD40 antibody, and 220 μg IRM1.Mice were sacrificed on each of days four, five, six, nine, and twelve.The spleens were removed from the sacrificed mice and homogenized. Thehomogenized cell suspensions were stained and analyzed as described inExample 1. When subjected to flow cytometry, ovalbumin-specific CD8⁺ Tcells (top) and ovalbumin-specific CD8⁺/CD44+ T cells (bottom) wereidentified and are shown in the upper right quadrant of each dot plot.The numbers in the upper right quadrant indicate the percentage of cellsin that quadrant. These data shown that the synergistic effect on CD8⁺ Tcell expansion observed in Example 1 also is observed with (a) adifferent CD40 agonist, and (b) full-sized ovalbumin protein as theantigen.

Example 3

Mice were immunized intravenously with 100 μg FGK4.5 anti-CD40 antibody+200 μg IRM1 and either (A) no peptide, (B) 100 μg ovalbumin peptide, or(C) 100 μg TRP2-ΔV peptide. At five days after the immunizations, thespleens were removed from the mice and homogenized. The homogenized cellsuspension was stained as in Example 1, except that the MHC tetramerreagent was prepared for detecting TRP2-ΔV-specific T cells. Whensubjected to flow cytometry, TRP2-ΔV-specific CD8⁺ T cells are shown inthe upper right quadrant of the dot plots shown in FIG. 3. The numbersin the upper right quadrant indicate the percentage of cells in thatquadrant. The data show synergistic expansion of antigen-specific CD8⁺ Tcells after stimulation with the combination of the anti-CD40 antibodyand an IRM with yet another antigen.

Example 4

Mice were immunized intravenously on day 0 with 100 μg ovalbuminpiptide+200 μg IRM1+100 μg of 1C10 anti-CD40 antibody. On day 28, themice were either (A) left unchallenged, (B) challenged intravenouslywith 100 μg ovalbumin peptide, or (C) challenged intravenously with 100μg ovalbumin peptide+200 μg IRM1. On day 33, the mice were sacrificed,the spleens removed and spleen cells homogenized. The homogenized cellswere stained and analyzed as described in Example 1. The data are shownin FIG. 4. The synergistic expansion of CD8⁺ T cells that occurs as aresult of immunizing with an antigen, a CD40 agonist, and an TLR agonist(shown in Example 1) generates a pool of long-lived CD8⁺ memory T cellsthat can be reactivated by treatment with IRM and the antigen, shown in(C).

Example 5

Mice were immunized intravenously as indicated in Table 2. At five days,the mice were sacrificed, spleens harvested, and the cells homogenized,stained, and analyzed as in Example 1. The data are shown in FIG. 5. Thenumbers in the upper right quadrant indicate the percentage of cells inthat quadrant.

TABLE 2 Immunization combinations for Example 5 Sample 3 mg ovalbumin100 μg CD40 agonist Stimulus A + + none B + − none C + + 50 μg CpG D + −50 μg CpG E + + 30 μg LPS F + − 30 μg LPS G + + 50 μg PolyIC H + − 50 μgPolyIC I + + 200 μg IRM1 J + − 200 μg IRM1

Example 6

Mice were challenged intradermally on day 0 with 1×10⁵ melanoma B16ovatumor cells in PBS (Kedl et al. PNAS 98(19):10811-10816). On day 7, themice were immunized with either (A) 100 μg ovalbumin peptide, (B) 100 μgovalbumin peptide+200 μg IRM1, or (C) 100 μg ovalbumin peptide+200 μgIRM1+100 μg 1C10 anti-CD40 antibody. On day 21, the mice were sacrificedand the tumors were measured in two dimensions by caliper. Data areshown in FIG. 6. Immunization with antigen, IRM and CD40 agonistresulted in slower tumor growth than immunization with IRM alone.

Mice also were challenged as described above, and immunized as describedabove except that IRM2 was substituted for IRM1. The results observedusing IRM2 in place of IRM1 were similar to the results observed usingIRM1.

Example 7

Mice were challenged with tu1×10⁶ cell equivalents (CE) (A) tumorlysate, (B) 1×10⁶ CE tumor lysate+200 μg IRM1, (C) 1×10⁶ CE tumorlysate+100 μg FGK4.5 anti-CD40 antibody, or (D) 1×10⁶ CE tumorlysate+200 μg IRM1+100 μg FGK4.5 anti-CD40 antibody. Tumor sizes weremeasured on the mice by caliper on days 14 and 20. The data are shown inFIG. 7. Immunization with the combination of IRM and anti-CD40 agonistsresulted in slower tumor growth than immunization with IRM alone or CD40agonist alone.

Example 8

Mice were intraperitoneally injected on day 0 with 500 μg ovalbumin, 50μg CD40 agonist (FGK4.5), and either 500 μg IRM3, 200 μg IRM4, 800 μgIRM5, 800 μg IRM2, or no IRM (control). On day 6, the mice weresacrificed and spleen cells were harvested and analyzed as described inExample 2. FIG. 8 shows the average percentage of CD8⁺ T cells observedin each group of mice (n=3 for each group). Synergistic expansion ofCD8⁺T cells is demonstrated using CD40 agonist in combination withdifferent IRM compounds.

Example 9

Mice were immunized on day 0 with 1 mg ovalbumin, 200 μg IRM1, andeither 200 μg CD40 ligand (FGK4.5), 200 μg 4-1BB ligand (anti-mouse4-1BB antibody, clone 17B3, eBioscience, San Diego, Calif.), or no TNF/Ragonist (control). On day 6, the mice were sacrificed and spleen cellswere harvested and analyzed as described in Example 2. The results areshown in FIG. 9. Synergistic expansion of CD8⁺ T cells is demonstratedusing IRM1 in combination with different TNF/R agonists.

Example 10

On day 0, a set of wild-type mice (B6/129 Fl, Taconic, Germantown, N.Y.)and a set of IFNαβ receptor knockout mice (National Jewish Medical andResearch Center, Denver Colo.) were injected intraperitoneally with 100μg SIINFEKL peptide, 50 μg FGK45 (CD40 agonist), and either (a) nothing(CD40 only), (b) 100 μg IRM1 (+TLR7), (c) 50 μg poly IC (+TLR3), 100 μgCpG (+TLR9), 30 μg LPS (+TLR4), or 25 μg MALP-2 (+TLR2). On day 6, themice were sacrificed and spleen cells were harvested and analyzed asdescribed in Example 2.

FIG. 10 shows the percentage of tetramer⁺T cells generated in wild-typeand IFN knockout mice after immunization of mice with immunostimulatorycombinations and, therefore, the IFN dependency of the synergisticimmune response when induce by immunostimulatory combinations thatinclude agonists of various TLRs.

Example 11

A set of wild-type mice (B6/129 Fl, Taconic, Germantown, N.Y.) and a setof IFNαβ receptor knockout mice (National Jewish Medical and ResearchCenter, Denver Colo.) were injected intraperitoneally with 50 μg FGK45(CD40 agonist) on day 0. Four hours later, the mice were injectedintravenously with 100 μg SIINFEKL alone, or with either 100 μg IRM1(TLR7 agonist), 25 μg MALP-2, 50 μg Pam3cys (Alexis Biochemicals, Corp.,San Diego, Calif.), 100 μg Pam3cys, or 250 μg Pam3cys. On day 6, themice were sacrificed and spleen cells were harvested and analyzed asdescribed in Example 2. The results are shown in FIG. 11. Theinterferon-independent synergistic immune response observed when animmunostimulatory combination that includes MALP-2, a TLR2/6 agonist, isalso observed using an immunostimulatory combination that includesPam3cys, a TLR2 agonist.

The complete disclosures of the patents, patent documents andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. In case of conflict,the present specification, including definitions, shall control.

Various modifications and alterations to this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention. Illustrative embodiments and examples areprovided as examples only and are not intended to limit the scope of thepresent invention. The scope of the invention is limited only by theclaims set forth as follows.

1. An immunostimulatory combination comprising: at least one TLR agonistand at least one TNF/R agonist, each in an amount that, in combinationwith the other(s), is(are) effective to synergistically increase asubject's immune response to a desired antigen, wherein the at least oneTNF/R agonist comprises a 4-1BB agonist.
 2. The immunostimulatorycombination of claim 1 wherein the at least one TLR agonist is anagonist of at least one of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR8, TLR9, TLR10, or any combination of any of the foregoing.
 3. Theimmunostimulatory combination of claim 2 wherein the TLR agonistcomprises an IRM compound or an agonist of TLR2, or comprises MALP-2,LPS, polyIC, or CpG.
 4. (canceled)
 5. The immunostimulatory combinationof claim 3 wherein the IRM compound comprises an imidazoquinoline amine,a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a1,2-bridged imidazoquinoline amine, a 6,7-fusedcycloalkylimidazopyridine amine, an imidazonaphthyridine amine, atetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, athiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridineamine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.6. (canceled)
 7. The immunostimulatory combination of claim 1 whereinthe at least one TNF/R agonist further comprises an agonist of CD40ligand, OX40 ligand, CD27, CD30 ligand (CD153), TNFα, TNF-β, RANKligand, LT-α, LT-β, GITR ligand, LIGHT and/or further comprises anagonist of CD40, OX40, 4-1BB, CD70 (CD27 ligand), CD30, TNFR2, RANK,LT-BR, HVEM, GIFR, TROY, or RELT. 8-9. (canceled)
 10. Theimmunostimulatory combination of claim 1 wherein the at least one TNF/Ragonist comprises an agonistic antibody.
 11. A method of inducing asynergistic antigen-specific T_(H1) immune response and/orsynergistically activating CD8⁺ T cells specific to a desired antigenand/or elicit a synergistic cell-mediated immune response specific to adesired antigen in a subject with a disease condition associated withcells which express said desired antigen comprising: co-administering tothe subject at least one TLR agonist and at least one TNF/R agonist,each in an amount that, when in combination with the other, is effectiveto induce a synergistic T_(H1) immune response and/or synergisticallyactivating CD8⁺ T cells specific to a desired antigen, wherein the atleast one TNF/R agonist comprises a 4-1BB agonist. 12-15. (canceled) 16.The method of claim 11 which further includes the administration of adesired antigen against which said synergistic T_(H1)immune response iselicited and/or to which antigen said activated CD8⁺ T cells arespecific. 17-18. (canceled)
 19. The method of claim 11 whereinactivating CD8⁺ T cells comprises expansion of CD8⁺ effector T cellsand/or generating antigen-specific CD8⁺ memory T cells. 20-26.(canceled)
 27. The method of claim 11 wherein the at least one TNF/Ragonist comprises an agonistic antibody.
 28. The method of activating orexpanding antigen-specific memory CD8⁺ T cells and/or generatingantigen-specific CD8⁺ memory T cells of claim 19, wherein the subjecthas had prior exposure to the desired antigen. 29-42. (canceled)
 43. Themethod of claim 11 wherein the condition comprises a neoplastic disease.44-45. (canceled)
 46. The method of claim 11 wherein the conditioncomprises an infectious disease. 47-48. (canceled)
 49. Theimmunostimulatory combination of claim 1, which further comprises adesired antigen against which said synergistic immune response iselicited. 50-56. (canceled)
 57. The immunostimulatory combination ofclaim 49 wherein the antigen comprises a tumor antigen, a viral antigen,a bacterial antigen, or a parasitic antigen.